T-cells, including cytotoxic T-lymphocytes (CTLs), are a critical component of effective human immune responses to tumors, viral infections and other infectious diseases. T-cells destroy neoplastic or virally infected cells through recognition of antigenic peptides presented by MHC class I molecules on the surfaces of target cells. Activation of T-cells is dependent upon coordinate signaling through antigen receptors and costimulator receptors on T-cell surfaces. Many mechanisms contribute to the escape of tumor cells and virally infected cells from immune surveillance. One of the mechanisms is that these cells lack the costimulatory molecules required for T-cell activation. Immunotherapeutic strategies have been developed that are predicated upon expressing costimulators on tumor cell, and other antigen-presenting cell, surfaces.
Professional antigen-presenting cells (APCs), by virtue of the surface costimulatory molecules, are geared towards potent T-cell activation. APCs can be converted into deletional APCs, or "artificial veto cells", by expressing coinhibitors at their surfaces. This is discussed, for example, in U.S. Pat. Nos. 5,242,687; 5,601,828; and 5,623,056. Such coinhibitors bind to coinhibitor receptors on cells, leading to T-cell inactivation.
One approach for expressing costimulators and coinhibitors on APCs, such as tumor cells, is gene transfer. When used for APC and tumor cell engineering, gene transfer techniques have shortcomings. For example, APCs, including tumor cells, are often poorly transfectable. In addition, transfection proceedings are cumbersome and time-consuming. Furthermore, expressing more than a costimulator (or coinhibitor) is difficult. These and other issues have impeded the widespread application of gene therapy for APC and tumor cell engineering.
Protein transfer offers a number of advantages over gene transfer for engineering APCs and other cells. These advantages include the ability to modify poorly transfectable cells (for example, biopsy-derived tumor cells), the simplicity of expressing multiple proteins on the same cell surface, and the relative ease and rapidity of the procedure. The successful use of recombinant GPI-modified costimulator and MHC protein derivatives for protein transfer has been reported. (See, Brunschwig, et al. J. Immunol., 155:5498 (1995); McHugh, et al; Proc. Natl. Acad. Sci. USA, 92:8059 (1995); and McHugh, et al. Cancer Res., 59:2433 (1999)). A shortcoming of the GPI protein transfer strategy, however, resides in scaling up the purification of GPI proteins from membranes of transfected cells.
Kim and Peacock, J. Immunol. Methods, 158:57 (1993), report the use of palmitate-conjugated protein A for coating cells with artificial receptors which facilitate intercellular interactions. More specifically, a method is reported for attaching an antibody onto the surface of a cell using palmitated protein A. The article does not teach use of a lipidated protein for attachment of anything other than an antibody to a cell. As such, their modified cells serve only as artificial receptors for antigens.
Phillips et al., Immunity, 5:163-172 (August, 1996) report the preparation of a fusion protein using a CD8 leader segment, the Fc domain, of immunoglobin and the ectodomain of a type II membrane protein, CD94. The present transfer methods are applicable to both type I and type II proteins and are neither taught nor suggested in the article.
Darling, et al., Gene Therapy, 4(12):1350-60 (December, 1997) report the use of a biotin/avidin-based system for protein transfer. This method involves biotinylation of the target cell, attachment of an avidin group to the protein to be transferred, and combining the biotinylated target cell and the avidin-tagged protein. This method has significant limitations, including its dependence on covalent modifications that could perturb multiple proteins on cell surfaces.
There remains a need, therefore, for methods of efficient and quantitative transfer of proteins and peptides to cells. A further need is to provide such methods in which immunoregulatory molecules that retain their function can be attached to cells of interest.